v-SNARE composition distinguishes synaptic vesicle pools.

1Department of Neurology, University of California, San Francisco School of Medicine, San Francisco, CA 94143, USA.

Abstract

Synaptic vesicles belong to two distinct pools, a recycling pool responsible for the evoked release of neurotransmitter and a resting pool unresponsive to stimulation. The uniform appearance of synaptic vesicles has suggested that differences in location or cytoskeletal association account for these differences in function. We now find that the v-SNARE tetanus toxin-insensitive vesicle-associated membrane protein (VAMP7) differs from other synaptic vesicle proteins in its distribution to the two pools, providing evidence that they differ in molecular composition. We also find that both resting and recycling pools undergo spontaneous release, and when activated by deletion of the longin domain, VAMP7 influences the properties of release. Further, the endocytosis that follows evoked and spontaneous release differs in mechanism, and specific sequences confer targeting to the different vesicle pools. The results suggest that different endocytic mechanisms generate synaptic vesicles with different proteins that can endow the vesicles with distinct properties.

VAMP7-pHluorin shows less response to electrical stimulation than VGLUT1-pHluorin

VAMP7-pHluorin-IRES2-synaptophysin(syp)-mCherry was transfected into primary hippocampal neurons, and imaged at 12-14 days in vitro. (A) Using NH4Cl (50 mM) to reveal the entire intracellular pool of pHluorin fusion, VAMP7-pHluorin and syp-mCherry partially colocalize. Synaptic boutons were selected for analysis on the sole basis of syp-mCherry fluorescence. (B) Response of VAMP7-pHluorin at individual boutons indicated in (A) to field stimulation at 10 Hz for 60 s, followed by NH4Cl. (C) Normalized to total internal pHluorin detected in NH4Cl, VAMP7 (red circles) shows more quenching at low external pH (5.5) than VGLUT1 (black circles), indicating a high surface fraction of VAMP7 (see also Figure S1C,D). In addition, VAMP7-pHluorin responds more weakly to stimulation than VGLUT1-pHluorin, and the recovery in fluorescence after stimulation is both delayed and incomplete. Since the VAMP7-pHluorin signal persistent after stimulation can be quenched by external MES buffer at pH 5.5, it reflects a delay in endocytosis rather than acidification. n=144 boutons from 3 coverslips for each construct (D) Analyzed by both cumulative frequency distribution and a direct comparison of the means (inset), the time constant for endocytosis after stimulation (τendo) is significantly slower for VAMP7-pHluorin than for VGLUT1-pHluorin. ***, p < 0.001; n = 5 coverslips containing a total of 289 boutons for VGLUT1 and 102 boutons for VAMP7. See also Figure S1.

(A to D) Hippocampal neurons transfected with VGLUT1-, VAMP2- or VAMP7-pHluorin were stimulated at 10 Hz for 60 s in the presence of bafilomycin to identify the recycling pool of vesicles, then treated with 50 mM NH4Cl to reveal the intracellular pool. Panel (A) shows the average traces of 142 boutons from 3 coverslips. (B) Relative to VAMP7, a substantially larger proportion of VGLUT1 and VAMP2 responds to stimulation. **, p < 0.01; ***, p < 0.001 (C) A histogram shows the distribution of recycling pool sizes for individual boutons expressing pHluorin fusions to VGLUT1, VAMP2 and VAMP7. n=9 coverslips containing a total of 438 boutons for VGLUT1, 473 for VAMP2 and 487 for VAMP7 (D) Recycling pool size shows no correlation with total pHluorin expression, and differs between VGLUT1 and VAMP7 over a range of expression levels. The data indicate mean ± SEM for individual coverslips containing 50-100 boutons each. See also Figure S2.

(A) Hippocampal neurons expressing VAMP7-pHluorin were loaded with FM4-64 by stimulation at 10 Hz for 60 s, washed for 10 min, and destained at 10 Hz for 120 s. Boutons with or without VAMP7-pHluorin show no difference in FM4-64 dye loading and unloading (τcontrol = 51.7 ± 1.0s and τvamp7 = 50.1 ± 0.9s). n = 89-90 boutons from 3 coverslips each (B) Cotransfection of untagged VAMP7 does not affect the recycling pool size of VGLUT1-pHluorin. n = 5 coverslips containing 269 boutons for without VAMP7 and 262 for with VAMP7 (C) Stimulating at 10 Hz in the presence of bafilomycin to exclude an effect of endocytosis, the time constant for exocytosis (τexo) shows no significant difference between VGLUT1- and VAMP7-pHluorin in terms of cumulative frequency distribution or a direct comparison of the means (inset). n = 6 coverslips containing 392 boutons for VGLUT1 and 296 for VAMP7 (D) The cumulative frequency distribution and mean (inset) of τexo do not differ between VGLUT1-pHluorin and VAMP7-pHluorin when stimulating in 0.5, 2 or 5 mM Ca++. n = 3 coverslips containing 120-146 boutons. Bar graphs in (B) through (D) indicate mean ± SEM. See also Figure S3.

(A) Hippocampal neurons were either not transfected or transfected with VAMP2- or VAMP7-HRP, then fixed and stained for HRP. In boutons expressing VAMP2-HRP and VAMP7-HRP, a subset of synaptic vesicles contain electron-dense deposit. Size bar, 200 nm. (B) The diameter of synaptic vesicles with electron-dense deposit does not differ from that of unlabeled synaptic vesicles, for neurons transfected with either VAMP2- or VAMP7-HRP. Bar graph indicates mean ± standard deviation. (C) The distribution of synaptic vesicle size shows no difference between those with and without deposit, for either VAMP2 or 7. n = 152-172 synaptic vesicles from 10-15 synapses for each condition. See also Figure S4.

(A) To measure spontaneous release, unstimulated hippocampal neurons expressing VGLUT1- or C were treated with the cell impermeant H+-ATPase inhibitor folimycin in the presence of TTX. The rate of spontaneous fluorescence increase was normalized to total internal fluorescence determined in NH4Cl. (B) Vesicles expressing VAMP7-pHluorin or VAMP2-pHluorin undergo a higher rate of spontaneous release than those expressing VGLUT1-pHluorin. **, p < 0.01; ***, p < 0.001; n = 6 coverslips containing a total of 394 boutons for VGLUT1, 332 for VAMP2 and 419 for VAMP7 (C) Hippocampal neurons expressed HA-tagged VGLUT1 or VAMP7 were first incubated with unlabeled mouse anti-HA antibody to block protein already at the cell surface, then labeled with anti-HA conjugated to Alexa 488 by field stimulation at 10 Hz for 2 min or spontaneous uptake in 0.5 μM TTX for 20 min. For control, the spontaneous uptake of HA antibody was measured over 2 minutes (also in TTX). The cells were then fixed, permeablized and immunostained for total HA protein using a secondary antibody conjugated to Alexa 635. (D) In neurons expressing VGLUT1-HA, stimulation for two minutes evokes much more uptake of HA antibody than observed spontaneously over 20 minutes. In contrast, spontaneous exocytosis of VAMP7 approaches that observed with stimulation. Size bar, 5 μm (E,F) The extent of cell surface delivery was normalized to total reporter expression. n = 10 fields containing a total of 500 boutons from 3 coverslips of each condition. Bar graphs indicate mean ± SEM. See also Figure S5.

(A) Hippocampal neurons expressing the pHluorin constructs indicated were either stimulated at 10 Hz for 60 s in folimycin (experiment 1, weaker colors) or incubated without stimulation in folimycin, CNQX and APV for 10 minutes, followed by stimulation at 10 Hz for 60 s (experiment 2, stronger colors). In both cases, NH4Cl was used to alkalinize intracellular compartments and reveal the total pHluorin fluorescence. The dashed lines indicate the size of the recycling pool determined in experiment 1, and the arrowheads indicate the predicted size if the two pools are independent. The fluorescence reached in experiment 2 exceeds that predicted if spontaneously released vesicles come entirely from the recycling pool, but falls short of that predicted if they were entirely independent. (B) The recycling pool size derived from experiment 1 (A) is shown as a stronger color, and the resting pool as weaker. The upper dashed line indicates the cumulative fluorescence derived from both spontaneous and evoked release. The lower dashed line indicates the residual fluorescence evoked by stimulation after 10 minutes of spontaneous release. The area between upper and lower dashed lines indicates the fluorescence derived from spontaneous release over 10 minutes. Spontaneous release derives from both resting and recycling pools. n = 6 coverslips with a total of 300 boutons per group (C) In hippocampal neurons expressing VGLUT1-pHluorin, the F-actin-destabilizing agent latrunculin A (5 μM) does not interfere with the synaptic vesicle exocytosis or endocytosis evoked by 10 Hz stimulation. n = 44 boutons for each condition (D) In unstimulated hippocampal neurons, folimycin and latrunculin A both increase the fluorescence of VAMP7-pHluorin to the same extent, and without any additive effect. n = 4 coverslips containing 137 boutons for control, 218 for folimycin alone, 237 for latrunculin A and 227 for folimycin and latrunculin A. Bar graphs indicate mean ± SEM.

(A) Domain organization of VAMP7-pHluorin with and without (VAMP7-ND) the N-terminal longin domain, and of VGLUT1-pHluorin with and without (VGLUT1-S3) the C-terminal polyproline motifs. (B) Transfected into hippocampal neurons and imaged as described in Figure 5A, VAMP7-ND-pHluorin exhibits significantly higher spontaneous release than wild type VAMP7-pHluorin. n = 7 coverslips containing a total of 361 boutons for wild type VAMP7 and 391 boutons for VAMP7-ND (C) Deletion of the longin domain increases the proportion of VAMP7 in the recycling pool as determined by stimulation at 10 Hz for 60 s in the presence of bafilomycin, and normalization to the signal in NH4Cl. n = 6 coverslips containing 305 boutons for wild type VAMP7 and 278 boutons for VAMP7-ND (D,E) Deletion of the C-terminal polyproline motifs increases the spontaneous release of VGLUT1 (D) (n = 5-6 coverslips containing a total of 380-680 boutons), without increasing the recycling pool size (E) (p = 0.3, n = 4-5 coverslips with 140-240 boutons) **, p < 0.01; ***, p < 0.001. See also Figure S6.